The aim of crop plant genetic engineering is to insert a gene (or genes) which improve what may be an already top performing plant variety whilst retaining the desirable genetic make up of the original plant. This technology can be integrated into traditional plant breeding programmes and apart from extending the potential genetic make-up of crops to include genes outside the normal gene pool, or synthetic genes, the process for introduction of new genetic information is rapid, much more precise and avoids the lengthy backcrossing programmes usually associated with the introgression of a gene via sexual crossing.
Genetic transformation is a relatively rare event and so most strategies for gene transfer into plants require the use of a transformation `marker` gene in order to `select` or `screen` for transformed plant material. Conventional marker genes are usually microbial in origin and include antibiotic resistance genes, herbicide resistance genes and genes coding for easily screenable enzymes. Transformed plant material are thus `selected` by an ability to grow in the presence of normally toxic levels of specific antibiotics or herbicides, or `screened` for by assaying for the expression of novel enzymatic activities not normally found in higher plant tissue.
The expression of marker genes is crucial only at phases of the gene transfer process when transformed cells/tissues/organs or whole plants are being selected or screened for. However, these marker genes are invariably under the control of strong, constitutive gene promoters (for example the Cauliflower Mosaic Virus 35S promoter) which drive marker gene expression in almost all tissue types at high levels throughout the life cycle of the plant. Although much effort has gone into the development of new marker genes, sufficient attention has not been paid to the specific control of marker gene expression. Improvement in the utility of marker genes, since their conception in 1983, has been very slight and has been concerned mainly with attempting to increase the levels of their expression.
One drawback of many marker genes is that they generally show poor expression in monocotyledonous cell types, particularly cereals, and one aim of this invention is the specific development of marker genes that function efficiently in monocot cells.
This invention relates to the improvement of transformation vectors and transformation procedures by engineering marker genes for their strong expression specifically in cell types which are targets for gene transfer. The invention also relates to ensuring or helping ensure strong gene expression in transformed cells at stages in the transformation process when selection is applied.
The invention further relates to enabling or improving the strong expression of genes in plant cell cultures, thereby enhancing the quantity or quality of the direct or indirect product of the genes. Direct products of genes are proteins (which term includes glycoproteins when the context so admits); indirect products of genes include non-proteins when the direct product is an enzyme.
The invention still further relates to the inducible expression of marker genes in transformed plant material to allow the application of more stringent selection conditions and to allow screening for a transiently inducible transformation marker phenotype in established transgenic shoots or transgenic plants at any stage in their life cycle.
To simplify plant breeding strategies it is preferable to obtain transgenic plants with only one copy of an inserted gene and accompanying selection marker gene. In many species, transgenic plants can only be recognised using high concentrations of selective agents. A current problem is that transgenic plants recovered from transformation processes using high levels of selective agents often contain multiple copies of the transferred DNA. It is postulated that high levels of marker gene expression are required to obtain transformants in this situation. A further aim of this invention therefore relates to the specific engineering of marker genes to drive high expression levels in transformed plant cells to allow transformants to be generated with a minimal number of copies of the transferred DNA.
The expression of transformation marker genes is not a required or indeed desirable feature of any improved plant variety. High level marker gene expression is common in transgenic plants. In commercially grown crops this gene expression represents a drain on plant resources to manufacture unwanted proteins and may result in yield depression. Moreover, the presence of large amounts of plant material containing particularly enzymes which inactivate antibiotics normally used to treat human and animal infections is certainly an undesirable feature of most foodstuffs currently derived from genetically engineered plants. Thus, a further aim of this invention is the manipulation of marker gene expression to minimise as much as possible marker gene products in commercially grown crop plants.